Background
Chronic pelvic pain (CPP) is a common symptom of endometriosis. Women with endometriosis are also at a high risk of suffering from anxiety, depression, and other psychological disorders. Recent studies indicate that endometriosis can affect the central nervous system (CNS). Changes in the functional activity of neurons, functional magnetic resonance imaging signals, and gene expression have been reported in the brains of rat and mouse models of endometriosis. The majority of the studies thus far have focused on neuronal changes, whereas changes in the glial cells in different brain regions have not been studied.
Methods
Endometriosis was induced in female mice (45-day-old; n = 6–11/timepoint) by syngeneic transfer of donor uterine tissue into the peritoneal cavity of recipient animals. Brains, spines, and endometriotic lesions were collected for analysis at 4, 8, 16, and 32 days post-induction. Sham surgery mice were used as controls (n = 6/timepoint). The pain was assessed using behavioral tests. Using immunohistochemistry for microglia marker ionized calcium-binding adapter molecule-1 (IBA1) and machine learning “Weka trainable segmentation” plugin in Fiji, we evaluated the morphological changes in microglia in different brain regions. Changes in glial fibrillary acidic protein (GFAP) for astrocytes, tumor necrosis factor (TNF), and interleukin-6 (IL6) were also evaluated.
Results
We observed an increase in microglial soma size in the cortex, hippocampus, thalamus, and hypothalamus of mice with endometriosis compared to sham controls on days 8, 16, and 32. The percentage of IBA1 and GFAP-positive area was increased in the cortex, hippocampus, thalamus, and hypothalamus in mice with endometriosis compared to sham controls on day 16. The number of microglia and astrocytes did not differ between endometriosis and sham control groups. We observed increased TNF and IL6 expression when expression levels from all brain regions were combined. Mice with endometriosis displayed reduced burrowing behavior and hyperalgesia in the abdomen and hind-paw.
Conclusion
We believe this is the first report of central nervous system-wide glial activation in a mouse model of endometriosis. These results have significant implications for understanding chronic pain associated with endometriosis and other issues such as anxiety and depression in women with endometriosis.
Graphical Abstract
Prenatal allospecific tolerance hinges on the developmental selection of NK cells expressing a friendly phenotype that is not cytotoxic to allogeneic donor cells. We wondered if prenatally educated friendly NK cells influenced allospecific T cell responses as an additional mechanism supporting prenatal tolerance. Therefore, we hypothesized that prenatally educated friendly NK cells exert suppressive effects on the induction of allospecific T cell responses.
To challenge this hypothesis, we examined the suppression potential of prenatally educated friendly NK cells through gain and loss of function experiments in stable prenatal Balb/c into B6.Thy1.2 chimeras (engrafter mice). We first depleted allospecific NK cells in host engrafter mice and then adoptively transferred Thy1.1+ naïve responder T cells along with CFSE-labeled allogenic or syngeneic target cells into the engrafter hosts. We found that the depletion of allospecific Ly49D+ NK cells in engrafter mice increased Thy1.1+ T cell alloimmunity in vivo. Conversely, adoptive transfer of friendly NK cells suppressed the induction of allospecific T cell responses in host mice. Furthermore, cultured friendly NK cells from engrafter mice showed stronger suppression on the induction of T cell alloimmunity in vitro than that of naïve mice. Lasltly, NK cell-mediated suppression of allospecific T cells required cell contact.
From these results, we conclude that prenatally educated friendly NK cell exert enhanced suppressive effects on T cell alloimmunity in vivo and in vitro. Depending on the context of their prenatal education, NK cells can differentially influence T cell responses in prenatal chimeras.
Allospecific NK cell hyporesponsiveness develops after in utero hematopoietic cellular transplantation (IUHCT) in mice who exhibit stable long-term engraftment. Conversely, allospecific NK cell responses are normal in mice that reject their graft. We hypothesized that these alternate NK cell responses also differentially influence T cell responses after IUHCT.
To challenge this hypothesis, we compared the helper or suppression potential of prenatally educated NK cells between prenatal recipients with stable engraftment (engrafter mice) and littermates that rejected their graft (rejecter mice) in a Balb/c --- > a B6 model of IUHCT. Allospecific NK cells from engrafter, rejecter, or naïve mice were co-cultured with naïve responder T cells (B6) and irradiated allogeneic target cells (Balb/c) in an in vitro IFN-g assay. Intracellular production of IFN-g was measured by flow cytometry of the naïve responder T cells after 66 hrs of co-culture.
We found that responder T cells displayed increased IFN-gamma production when co-cultured with allospecific NK cells from rejecter mice compared to co-culture with allospecific NK cells from naive control mice. Conversely responder T cells displayed decreased IFN-g production when co-cultured with allospecific NK cells from engrafter mice compared to co-culture with NK cells from naïve control mice.
From these results, we conclude that allospecific NK cells can influence the allospecific response of T cells in vitro depending on the context of their prenatal education. Further studies are needed to determine if similar mechanisms support engraftment or rejection in vivo.
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